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The CRDF Rootstock Field Day will begin at 9:30 a.m. The field trial at Peace River Packing Company consists of one common Valencia clone on the following rootstocks: US-812, US-897, US-942, UFR-2, UFR-3, UFR-4, UFR-16, and Carrizo. This site is one of the large multi-location, replicated, three year old trials of Valencia on six candidate HLB-tolerant rootstocks compared to standard trees on US-812. Other locations are on the Ridge (Blumberg) and Flatwoods (LaBelle). Horticultural performance will be discussed along with the distribution of summary data and maps. Attendees will be free to explore the location. Drs. Jude Grosser and Kim Bowman will be speaking. There will be one CEU in crop management for CCAs. For more information, please contact Chris Oswalt, 863-519-1052, wcoswalt@ufl.edu

The Status of Orange Juice Supply

By Marcos Fava Neves

The newest estimate from the U.S. Department of Agriculture for the 2017–18 season shows Florida with only 46 million boxes of oranges coming from 50 million trees, which represents 33 percent lower production than the 2016–17 season. High droppage and small fruits are also expected.

Rabobank estimates Florida’s juice production at 235,000 tons [frozen concentrated orange juice (FCOJ) equivalent] in 2017–18 and 250,000 tons in 2018–19. Total output in 2016–17 was 390,000 tons. The orange juice chain is fortunate that Brazil has 385 million boxes and is able to supply the world markets. However, if a huge price increase occurs, that could further damage juice consumption.

BRAZILIAN EXPORTS UP
Since the beginning of the current season, exports from Brazil are 25 percent higher in volume and 32 percent higher in income. Because of Hurricane Irma’s damage to Florida citrus, approximately 30 million boxes now have to be crushed and diverted to the United States, complicating logistics.

Rain came back in Brazil, but extreme weather created problems for the first bloom. The effects on the 2018–19 crop are unknown at this point, but will be announced on May 10. Rabobank estimates Brazilian 2017–18 production at 1.2 million tons of FCOJ, including approximately 335 million boxes for crushing and 50 million for the internal market. Rabobank’s 2018–19 projection of 950,000 tons is a little bit more conservative. The estimate also shows a demand decline of 4 percent per year in the next two years.

BIG BRAZILIAN CROPThe new Fundecitrus estimate published on Dec. 11 for the current crop in the Brazilian orange belt is 385 million boxes, coming from roughly 175 million trees. It is 57 percent higher than last year, nearly 3 percent larger than the last estimate, and 5.69 percent greater than the initial estimate published in May.

Fruits have bigger weight due to above-average rains at the beginning of the crop and better treatment of the orchards. As of mid-December, early-season oranges are almost all harvested, mid-season oranges are 90 percent picked, and late-season oranges are approximately 60 percent harvested. Factories are working at full capacity to process this large crop.

MARKET OUTLOOKSome recovery of the very low and dangerous juice stock levels will be seen, but we will still be dependent on a good 2018–19 crop to bring normality back. Current prices are good for farmers.

I expect juice prices in Europe to be near $2,400 to $2,500 (bulk FCOJ price per metric ton) for the coming months. If Brazil’s current crop (July to June) was as small as last year’s crop, combined with the problems of the Florida crop (October to September), there would be a huge shortage, causing terrible problems for the world’s orange juice supply. With Brazil’s large expected production, fortunately this is not the case.

Marcos Fava Neves is a professor at the University of São Paulo (Brazil), international adjunct professor at Purdue University (Indiana) and author of “The Future of Food Business” (World Scientific, 2014).

* Courtesy of SE AgNet Media

WINTER HAVEN – Florida citrus officials greeted the Environmental Protection Agency’s recent decision to extend use of three bactericides positively, although their effectiveness in moderating the effects of citrus greening remain unproven.

“It’s a good thing we got it so we can re-evaluate it,” said John Barben, an Avon Park grower and president of Florida Citrus Mutual in Bartow, the growers’ trade group. “It’s a tool in our toolbox, and we need all the tools we can get.”

The EPA had long approved use of the three bactericides — two brands of oxytetracycline and a streptomycin compound — for stone fruit and other crops but not for citrus. Research sponsored by the Lake Alfred-based Citrus Research and Development Foundation Inc., the chief scientific agency in the fight against greening, showed the bactericides could diminish the effects of greening, including increased levels of pre-harvest fruit drop and smaller fruit.

In March 2016, the agency approved a one-year “Section 18” special-use exemption for citrus. The EPA on Jan. 17 approved a third year of the exemption.

“I applaud the EPA’s decision to once again allow the use of antimicrobials on diseased citrus trees,” said U.S. Sen. Marco Rubio in a press statement following the decision’s announcement. “Our iconic citrus industry has long battled greening, and especially following the devastation of Hurricane Irma, this announcement is greatly appreciated. However, Florida agriculture is still in desperate need of disaster relief to help replace lost trees, rehabilitate flooded groves, and assist beleaguered farmers. I continue to work with my colleagues in Congress to secure this much-needed support for Florida farmers.”

Barben said he and many other citrus growers began using the bactericides right after the 2016 announcement. He said he plans to continue using the bactericides this season but at reduced rates because of the economic impact from the fruit lost to Irma.

“It’s an expensive spray,” he said. “These days you have to be careful what you put out there. You have to consider how much bang for the buck you get.”

Harold Browning, the chief operating officer at the Citrus Research Foundation, welcomed the EPA announcement, but he acknowledged the scientific data on the bactericides’ effectiveness is “noisy,” meaning there’s conflicting data.

Irma has complicated the ongoing effort to assess their effectiveness, he said. That’s because the storm dramatically reduced citrus tree yields, the number of fruit harvested per tree, one of the chief bactericide benefits.

“We’ll continue to take yield data, but there will always be an asterisk because 20 to 40 to 60 percent of the fruit was lost to Irma,” Browning said. “Although we’ll have the data, we’ll be unable to make inferences.”

Barben saw a 12 percent decline in the harvest of early and mid-season orange varieties, picked November to March, this season, he said. But he counted about one box of fruit per tree on the ground because of Irma, which would have meant an increased harvest but for the storm.

There was some good news about bactericides from Tom Jerkins, president of Premier Citrus in Vero Beach, one of the state’s largest growers, Browning said.

In a presentation earlier this week at the Florida Citrus Show in Fort Pierce, Jerkins reported a 15 percent yield increase in his 2016-17 crop of Valencia oranges, picked from March to June, he said. He had been using bactericides since 2016.

The increase is similar to results in some other studies, Browning said.

“There seems to be come convergence” on the 15 percent increase, he said.

In an email to The Ledger, Jerkins declined to make the presentation available. He could not be reached on Friday to comment.

Citrus officials hope this will be the last EPA exemption necessary because the agency is considering a request by the bactericides’ manufacturers to add citrus to the label of approved uses.

EPA officials have indicated they will have a decision on full approval of the bactericides for citrus in the first half of this year, Browning said. However, that date could be extended.

Greening is a bacterial disease that eventually kills a citrus tree. Infected trees produce fewer and smaller fruit, and they tend to drop what fruit they do produce before harvest.

The FAST TRACK model was developed as a means of making promising experimental UF/IFAS fresh citrus varieties available to nurseries and growers for trial and possible expedited commercial production. This innovative program engages growers and nurseries much earlier in the process, enabling participants to gain experience with the selections and make determinations of horticultural characteristics and performance as well as market acceptance of the final product.

To date, three suites of FAST TRACK citrus varieties have been introduced to the Florida citrus industry, resulting in small-scale diverse trial sites scattered throughout the traditional citrus production areas, as well as the northern border counties.

The beauty of the FAST TRACK model is the speed with which these selections have been moved through the system and into real-world evaluations.

The challenges are:
• Nurseries are having to rapidly learn how to grow these selections. Each behaves a little differently in the nursery and the process entails much more than budding trees for rapid increase of plant material.
• Trees are planted in a wide range of locations and conditions. Soil, water, temperature, and other variables factor into tree performance.
• A range of rootstocks were used with each scion. Thus far, through three suites of FAST TRACK releases, there are 127 scion/rootstock combinations in the field. Some were planted quite extensively, and some were planted in very small quantities.
• The trials are not of a scientific design. Growers ordered the combinations they wanted in the quantities desired. Grove layout and design differs from farm to farm, as do production practices.

Mind Your Material
Diversity is both a friend and foe in the evaluation of FAST TRACK selections. The program is designed to enable growers to plant new material and make their own determination of performance and marketability. Trial sites are for grower observations not research-oriented data collection.

Notwithstanding, it also is important that we make every effort possible to leverage these diverse trial sites to inform future planting decisions. Growers wishing to plant these selections in the future will have the reasonable expectation that information was compiled during the trial stage and early days of commercial production. Some FAST TRACK selections will not move into commercial production. Some will fail, while others may have niche market or dooryard potential. However, growers will need some basis for planting those FAST TRACK selections that prove to have commercial utility.

Observe and Report
The primary source of information related to FAST TRACK selections originates from trial report forms. When executing a FAST TRACK Tier I grower agreement, growers commit to periodically completing trial report forms and sharing information related to their trial plantings. Tier II growers share this obligation. However, after some discussion, New Varieties Development & Management Corp. (NVDMC), Florida Foundation Seed Producers Inc., and the UF/IFAS Plant Improvement team concluded there is added value in visiting the FAST TRACK trial sites, making observations about tree performance/health and production. When appropriate, it would be desirable to record information about fruit characteristics, productivity, and/or fruit quality.

After a considerable search, the NVDMC Board of Directors elected to contract with a crop scouting and monitoring company. If you are a FAST TRACK grower with trees from Suites I-III, expect a visit this season from On Point Ag. Growers will be contacted in advance, and an appointment will be established for a trial site visit. Grove maps are most helpful for these visits, as are any notes or records related to grove care, pruning, nutrition, bloom, fruit set, disorders, tree health, stress, etc. The field scout will take some measurements, snap a few photos, and ask a few questions (having an involved party present to answer questions is most appreciated). Everything gathered will be strictly confidential and will only be shared in a compiled general format.

No information from these visits will be associated with any particular grower. It is hoped as more visits are made, trends will begin to emerge that may prove helpful to growers, breeders, nurseries, and NVDMC.

This project may reveal that certain selections perform better in specific locations or conditions. We may learn that certain scion/rootstock combinations are superior and others are to be avoided. We may end up with more questions than answers or a general idea of what needs to be explored more extensively. This is but the first step in a process to glean meaningful information from small trial sites with tremendous geographic, horticultural, and operational diversity.

If you have FAST TRACK trees and would like to contact On Point directly, use onpointag@live.com to get on the calendar.

Do I Hear a Bingo?
‘Bingo’ is now in FAST TRACK Tier I trials and Tier II production. Though trees are still quite young, it is time to gather growers and nurseries (involved with this variety) for an exchange of information. NVDMC will host a ‘Bingo’ discussion opportunity at the UF/IFAS Citrus Research and Education Center on Feb. 20.

The last UF/IFAS variety display of the season will be held from 10:00 a.m. to 11:30 a.m. The ‘Bingo’ discussion is for registered growers and nurseries only and will take place in the Ben Hill Griffin Auditorium immediately following the display. This will be a working lunch. Box lunches are available for $10. Please RSVP to Lucy Nieves (lucy.nieves@ffva.com) and let her know if you would like a lunch.

Grafted orange trees showcased at the 100th anniversary of the UF/IFAS Citrus Research and Education Center may give the state’s citrus growers new reason to hope the industry can cope with the citrus greening crisis, UF/IFAS researchers say.

University of Florida scientists and Tropicana are teaming up to test promising new UF/IFAS-bred citrus varieties for greening tolerance and better-tasting juice. Tolerance means trees are infected and remain infected, but they still perform adequately to make a profit, UF/IFAS researchers say. Resistance means the trees fight off the infection – either reducing the infection to a very low level or getting rid of it completely.

“I’ve been out to the site twice in the past year and some of the rootstock-scion combinations just jump out at you with the lush green foliage and, more importantly, a large number of nice-sized fruit,” said UF/IFAS CREC Director Michael Rogers.

“What’s more, there are multiple large plots for each rootstock combination that allow us to really get a good idea of how these trees are holding up under greening,” Rogers said. Greening, also known as huanglongbing, or HLB, has impacted more than 80 percent of Florida’s citrus trees, according to UF/IFAS research. Rogers added: “From what I’ve seen, we’ve got some reasonably tolerant scion/rootstock combinations that growers should be taking a look at as short-term solutions to living with greening until true HLB-resistant trees are developed.”

Tropicana leases 14.5 acres for testing new selections including UF/IFAS-developed citrus cultivars and rootstocks in a commercial grove about 4 miles from the UF/IFAS Citrus Research and Education Center, said Jude Grosser, a UF/IFAS professor of horticultural sciences and a faculty member at CREC. UF/IFAS researchers have a long history of working with Tropicana, Grosser said.

“This trial is providing strong evidence that the combination of improved scion genetics, improved rootstock genetics and optimized nutrition programs is the ticket for the immediate future,” Grosser said.

For example, the combination of UF sweet orange OLL-8 cultivar on UFR-4 rootstock looks exceptional for both yield and tree health, Grosser said.

“These clones are a good immediate-term solution to help us until resistant clones are available,” he said. “We are continuing to screen rootstock hybrids for HLB tolerance that can be transmitted to a grafted scion. So far, we have screened more than 10,000 hybrids, and we have identified a few truly promising selections. Citrus breeding is a continuum – we are constantly building on previous progress.”

The relationship between Tropicana and UF/IFAS goes back many years.

“They became interested in our sweet orange improvement work through research interactions and presentations made at trade shows and seminars,” Grosser said. “We began sending them fruit samples from promising selections for sensory analysis.”

Rocco Simonetta, who’s now retired from Tropicana, spearheaded the UF/IFAS-Tropicana Project Field trials – with the first two trials focusing primarily on sweet orange selections in our pipeline that might be good additions to their Not-From-Concentrate (NFC) portfolio, Grosser said. The current trial focuses more on the addition of new rootstocks as necessary to deal with HLB.

Cheryl Nagle of Tropicana worked with Simonetta at the beginning, and she passionately followed up on building the relationship after Rocco’s retirement, Grosser said. She now is in charge of the trials.

“This is all truly a team effort,” he said.

Tropicana and UF/IFAS started their collaboration on the current project in June 2013, said Allison Drown, who manages the grove near Lake Alfred for Tropicana.

“We monitor tree health, fruit yield and juice quality,” Drown said.

Grosser commended Tropicana’s interest in testing many new UF/IFAS selections in their citrus trial.

“Tropicana has taken no shortcuts – they are employing state-of-the-art management of the trial,” said Grosser, who led a tour of the private groves at the Nov. 29 centennial celebration of the UF/IFAS CREC.

Tropicana and UF/IFAS breeders started with about 30 scion selections — or descendants of other varieties, Grosser said, but because of citrus greening, Tropicana decided to look at new and improved rootstocks as well, he said.

Citrus scientists use traditional plant grafting to put together combinations of new scions and rootstocks created in the breeding program.

In plant grafting, scientists call the upper part of the plant the scion, while the lower part is the rootstock. Grosser said he sees promise in about half a dozen scion-rootstock combinations in this trial, most of which are releases from UF/IFAS CREC.

Based on UF/IFAS data, UF/IFAS citrus breeders suggest new oranges, orange-like hybrids and rootstocks from the CREC citrus improvement program that they think Tropicana might be interested in for future use in its business, Grosser said.

“With our advice, Tropicana makes the final decisions on which scions and rootstocks are in the trial, and also the decisions on how to manage the trees,” he said. “We are currently working with Tropicana to develop a plan where we can work together to get good yield data this year.”

Sowing the Seeds of Hope for Florida Citrus

The Florida Department of Agriculture and Consumer Services Division of Plant Industry’s (FDACS DPI) Bureau of Citrus Budwood Registration has several significant duties. Among them is to maintain disease-free source material for Florida citrus nurseries, test to ensure the cleanliness of private nursery budwood sources, and supplement private nursery budwood supplies with cuttings from the foundation block in Chiefland.

In other words, the Bureau serves to encourage and enable nurseries to establish and maintain private budwood sources (scion and increase blocks), and can supply budwood to nurseries when private sources are insufficient.

Typically, budwood can be harvested from private nursery increase trees for 36 months. As a means of helping nurseries increase budwood faster, DPI is permitting nurseries to harvest wood from increase trees for 60 months without having to convert them to scion trees.

However, any trees that wish to be used in this process will have composite testing done at the 36-month period to allow their eligibility for the additional 24 months. This will enable the nurseries to keep these trees in budwood production longer, without having to cycle in new trees. This seems like a simple step, but it is yet another incremental improvement in the process and a means of addressing the pressing needs of industry.

A Numbers Game
There were 21.6% fewer total citrus tree propagations in the 2016-2017 season than the prior year. DPI’s budwood cutting decreased 22%. Sixty-four percent of the budwood used for propagations is now coming from private sources. Essentially, the system is performing as designed. Once better HLB solutions are available, there may still be some lag time in ramping up budwood quantities, but steps are being taken to improve preparedness. Strategically, DPI has been comprehensively studying the entire process of citrus tree production and how they might proactively tackle the next impediment to rapid industry expansion. It would be tragic to have access to better tools to combat HLB, experience a sharp increase in the demand for trees, and not have the ability to produce and supply. These discussions rapidly focused the conversation on rootstock seed availability.

The number of new rootstocks available to nurseries and growers has be rapidly escalating. Many growers are experimenting with a range of scion/rootstock combinations, while larger trial plantings are hoped to produce data that will further inform future planting decisions. Growers are sometimes forced to alter their planting plans as seed for the most popular (and hopefully tolerant) rootstocks (US 942, UFR-5, etc.) is in short supply. Seed availability from UF/IFAS Southwest Florida Research and Education Center (SWFREC), A.H. Whitmore Foundation Farm, and private sources is not meeting demand.

What about rootstock liners from tissue culture (TC)? Doesn’t that solve the problem? Relative to overall demand for citrus trees, TC liners are certainly coming online at just the right time, but more seed is needed. Liners from seed are less expensive for nurseries to produce, and locally produced seed has a higher germination rate than imported seed. Any project to increase rootstock seed availability would be complementary to escalating TC liner production. Seed will continue to be in short supply. TC production will help fill the gap until seed production catches up. There will always be nurseries and growers who prefer one over the other.

Prime Property to Grow On
The Bureau’s Chiefland budwood facility is located on Florida Forest Service property. This location has been ideal for its current purpose, as it is far enough from the coast to provide limited protection from coastal storms and is outside of the main citrus production areas. It just so happens that a 10-acre block of Florida Forest Service land is available (and has been donated) just south/southwest of the existing Budwood facility.

This property has been deemed suitable for a new seed block planting. Since the trees will be outdoors and exposed, it would not be ideal to have the seed block adjacent to the budwood screenhouses. This new seed block site is approximately 1 mile away from the current structures (as the crow flies). The new block will accommodate 40 to 50 rootstock varieties, with greater volumes of those currently in high demand.

The trees for the project will start their life at the FDACS LaCrosse Phase II Budwood Facility. Here, seed has been planted (enough to produce 2,000 trees) to serve as a root system for the seed source trees. Once the base rootstock trees are of sufficient size (it is anticipated that this will be February 2018), they will be grafted with budwood of the target commercial rootstock varieties. All of this will be done inside a clean, protected facility. The plants will then be transferred to the Chiefland block in August 2018. It should take approximately three to four years before fruit is available for seed harvest from these trees. Larger volumes of seed will be available in 2020-2021.

Most of the trees in the new seed block will be planted on X-639, but DPI is considering planting some seed trees from rooted cuttings so that production could be restored more quickly after an extreme cold weather event.

Stocking Up
The Florida Nursery, Growers and Landscape Association administered a grower survey in December to help determine the rootstock varieties that should be included in the new block. As more promising rootstocks are identified, they will be added to the DPI block, and eventually less successful selections will be replaced. All equipment to be used in the management of the new seed block will be dedicated to the farm to further minimize exposure to outside infection. Equipment is being sourced to enable DPI to perform the seed extraction on premises.

Funding for this project is being provided through the Citrus Health Response Program. This is a one-time funding source intended to address Florida’s declining rootstock seed production over the past seven years. HLB has reduced the volume of fruit on rootstock source trees and in some cases, the volume of seed per fruit. The seed block will be maintained using other funding sources moving forward. One additional staff position will be required for the project, but the seed block will be managed through the FDACS Chiefland office.

Though there are no guarantees, the industry continues to demonstrate its belief that better days are ahead and solutions will be found. Any actions that will enhance Florida’s capacity to recover are welcomed and appreciated; as is the forward thinking responsible for their implementation.

Progress on Engineering HLB-Tolerant/Resistant Citrus

By Manjul Dutt and Jude W. Grosser

Huanglongbing (HLB) threatens the survival of the Florida citrus industry. Both conventional cultivars and genetically engineered cultivars that can grow in the presence of HLB have been developed at the University of Florida’s Citrus Research and Education Center (CREC) in Lake Alfred. Among the two citrus improvement techniques, engineering citrus remains the fastest method for the improvement of an existing commercial cultivar and has been a key component in CREC’s overall citrus improvement strategy.

SYSTEMIC ACQUIRED RESISTANCEIn nature, plants are constantly attacked by pathogens; however, most attacks do not lead to infection. Plants resist getting infected with several invading pathogens by turning on their internal defense system, which is similar to the human immune system. This internal defense system is activated by a localized resistance reaction, which subsequently spreads throughout the plant. As a result, several protein molecules are produced inside the plant. These proteins, called the pathogenesis-related (PR) proteins, are ultimately responsible for preventing the invading pathogens from causing disease. The overall process of this immunization technique is called systemic acquired resistance (SAR).

Engineered citrus, which can manipulate the way in which these PR proteins are activated, can potentially keep the plant primed against infection and effectively combat diseases. This mechanism of induced defense can confer long-lasting protection against HLB.

Currently, a population of engineered sweet orange trees expressing the Arabidopsis NPR1 gene is being monitored for resistance to HLB. Several transgenic lines have been observed to provide durable resistance against HLB. Several lines have remained HLB-negative for over five years, although they have been planted at a site with >95 percent HLB infection (see Figure 1).The Arabidopsis (a mustard plant relative)-derived NPR1 gene plays an important role in priming the production and expression of PR proteins. This gene acts as a switch and aids in the activation of the PR proteins. CREC study has shown that citrus trees that are engineered with this gene have many activated disease-fighting PR genes.

Unfortunately, these transgenic Hamlin plants also contain the green fluorescent protein (GFP). GFP-expressing plants cannot be commercialized currently, as the GFP gene has not yet been approved for human consumption (although it comes from jellyfish that are consumed by humans). Selected sweet orange cultivars such as EV1, OLL-8, Valencia, Vernia and others are being retooled to express the Arabidopsis NPR1 and protect trees against HLB. These new trees are being produced without GFP.

Additional genes, such as the tobacco-derived SA-binding protein 2 (SABP2) or the citrus-derived β-1,3–glucanase (BG)gene, have also been inserted into citrus with promising results. The SABP2 gene is critical for the plant immune response; although BG is a PR gene, it may also reduce phloem plugging. Several transgenic rootstock lines expressing NPR1 have also been produced. These are now being grafted with commercial, non-GMO sweet orange scions for field-testing to determine if the improved tolerance can be transmitted to the scion.

GENE STACKINGGene stacking involves the expression of multiple genes in plants. Citrus is a long-lived perennial tree, and it is indeed possible that Candidatus Liberibacter asiaticus (CLas), the bacterium that causes the HLB disease, could mutate over time and become tolerant to the effects of a specific gene. This could result in the breakdown of disease resistance. Thus, the CREC team is engineering trees (scions and rootstocks) that contain two or more transgenes stacked in the plant that can back each other up. These plants are in the primary stages of evaluation, and their long-term ability to resist CLas is presently unknown.

This was done by enhancing the expression of the citrus flowering locus T (FT) gene. FT has been implicated in the induction of flowering in many plants. Like the NPR1 described before, FT can also travel through the plant’s vascular tissues. The hope is that an engineered precocious rootstock can stimulate the above-ground scion to flower in a much shorter time than naturally possible.

PRECOCIOUS CITRUS ROOTSTOCKS
Depending on the cultivar, citrus can remain juvenile for three to 10 years. This long juvenile phase hinders the evaluation of hybrids in the conventional breeding program as well as the engineered citrus developed from juvenile tissues. A reduction in juvenility can significantly reduce the assessment time for both, thus accelerating commercialization. Engineered Carrizo citrange rootstocks that flower within the first two years (Figure 2) have been developed.

This would enable rapid cycling through the breeding population to identify select lines that can be subsequently evaluated on a larger scale. Moreover, it would aid in the rapid evaluation of the CREC-engineered lines. In a preliminary test, a juvenile grapefruit clone grafted to one of the CREC-engineered Carrizo rootstock lines flowered only in its second flush, which was an exciting development.

CRISPR/Cas9All crop plants have evolved through the induction of mutations. Many navel orange and grapefruit cultivars are products of natural mutations. CRISPR/Cas9 is a tool that creates mutations in a plant’s DNA, but this process can be manipulated to occur much faster than that obtained through natural methods. CRISPR/Cas9 can target specific parts of a plant’s DNA and make necessary changes without affecting the rest of the plant.

In citrus, there is a potential to create an HLB-tolerant plant using this technology. Citrus contains a native NPR1 gene that is not very effective in protecting against HLB. A primary issue preventing its expression is the presence of other genes that suppress the function of this endogenous NPR1 gene.

The CREC research group has pioneered the cell culture and protoplast technologies that assist the endeavor to create targeted mutations in the citrus DNA. CRISPR/Cas9 technology is being utilized to target several negative regulators (proteins that suppress other useful proteins) of the SAR process and citrus NPR1 activity. It is expected that citrus plants would effectively combat HLB infection without the presence of these negative regulators. An added advantage is that these plants would be considered non-GMO (genetically modified organism), allowing for an easier commercialization process.

CONSUMER-FRIENDLY CITRUSTo address public perception issues that arise from the utilization of bacterial or viral components in the engineered plants and to have a more consumer-friendly appeal, a system has been developed that utilizes all plant-derived genetic elements coupled with a visual selection system using a citrus-derived anthocyanin gene (replacing the GFP above). The process involving the development of engineered plants is highly dependent on the ability of the researcher to select the genetically engineered cells from the background non-engineered cells.

Genetic markers such as the GFP are short pieces of DNA that visually indicate the presence of certain genes in an engineered plant. In contrast to the existing systems, a system has been developed that relies on the ability of cells to express anthocyanins in the initial stages of selection. This newly developed system also prevents the genetic marker gene from being expressed in the resulting engineered plants, but at the same time allows the expression of the beneficial gene(s) that allows the engineered plants to be protected from CLas.

The CREC team has developed anthocyanin, overexpressing, purple Mexican limes as a proof of concept during the evaluation of the anthocyanin system as a genetic marker. (Purple margaritas are just a side benefit!) The tropical and subtropical citrus plants do not produce anthocyanins, with the blood oranges producing anthocyanins only after a prolonged cold chill. While blood oranges can be grown in Florida, they lack the intense coloration.

IS GENETICALLY ENGINEERED CITRUS REALLY NEEDED?Anthocyanins are health-promoting compounds found in commonly consumed fruits such as blueberries, grapes and strawberries. CREC-developed plants offer proof that engineered citrus can result in the incorporation of an additional health-promoting component to the existing citrus portfolio (see Figure 3).

As with conventional breeding techniques, there are pros and cons to developing engineered citrus. Many believe that engineered citrus provides the best hope for a permanent solution to HLB.

Several questions remain unanswered. Will these CRISPR/Cas9 induced mutations have the desired effect? Will the engineered plants hold up against HLB over time? Will the stacked gene-containing plants have a better effect than plants containing a single gene in the long term, such as after 10 years in the field? Currently, the answers to all these questions are unknown. However, the possibility of viable options can be predicted for future citrus plantings — including conventionally bred, engineered trees or a combination of both — in Florida. Current results give hope that research is well on its way to winning the fight against HLB.

Manjul Dutt (manjul@ufl.edu) is a research assistant scientist and Jude Grosser (jgrosser@ufl.edu) is a research professor, both at the University of Florida Institute of Food and Agricultural Sciences Citrus Research and Education Center in Lake Alfred.